Cardiomyocyte cell cycle induction offers the potential for restoration of myocardial mass, and consequently contractile function, following cardiac injury. Considerable effort has thus been invested studying the degree to which cardiomyocytes can reenter the cell cycle and progress through cytokinesis in normal and injured adult mammalian hearts. Using a transgenic reporter system to identify cardiomyocyte nuclei in tissue sections in conjunction with continuous BrdU infusion, we have developed a digital imaging and analysis system which permits both quantitation and 3D anatomical mapping of cumulative cardiomyocyte S-phase activity across the entire heart. Using this system, we observed discrete clusters of cardiomyocyte S-phase activity in mice with permanent coronary artery ligation. We also observed very high rates of cardiomyocyte S-phase activity in the remote myocardium of mice with ischemia/reperfusion (I/R) injury. The studies proposed in this application will identify the underlying mechanistic basis for the differential cardiomyocyte cell cycle responses observed following myocardial injury. In Specific Aim 1, the variability in cardiomyocyte S-phase induction and cell cycle progression following permanent coronary artery ligation will be established and the resulting data sets will then be used for mathematical modeling with the goal of establish sampling criteria to quantitate total heart cardiomyocyte S-phase activity which takes into account these intrinsic anatomical variations. Other studies will determine if the observed clusters of S-phase activity arise from the clonal expansion of a subset of cardiomyocytes which retain the potential for cell cycle reentry. In Specific Aim 2, I/R injury will be performed in reporter mice maintained in an inbred genetic background to determine if the nature and/or degree of injury are responsible for high levels of cell cycle induction in the remote myocardium. Other studies will utilize informative backcrosses to determine the extent to which modifying genes can impact cardiomyocyte cell cycle reentry following I/R injury. In both Aims, the degree to which the S-phase positive cardiomyocytes progress through the cell cycle will also be quantitated. The proposed experiments will establish a 3D atlas of cardiomyocyte S-phase activity in response to commonly used and clinically relevant injury models, and will establish the degree to which increased levels of cardiomyocyte DNA synthesis contribute to polyploidization, multi-nucleation, and/or cardiomyocyte renewal. In addition, these experiments will characterize the impact of gender, genetic background and mode of injury on the magnitude of cardiomyocyte cell cycle reentry, as well as determine the consequences of natural variation in cardiomyocyte cell cycle activity on cardiac function post-injury. These data will provide useful insight for the development of interventional strategies with which to promote regenerative growth of the heart, as well as provide a comprehensive reference set for studies aimed at inducing cardiomyocyte renewal.